Dynamic behavior of GFP-CLIP-170 reveals fast protein turnover on microtubule plus ends

J Cell Biol. 2008 Feb 25;180(4):729-37. doi: 10.1083/jcb.200707203. Epub 2008 Feb 18.

Abstract

Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Binding Sites / physiology
  • COS Cells
  • Chlorocebus aethiops
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Mice
  • Mice, Transgenic
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Protein Binding / physiology
  • Protein Transport / physiology
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Time Factors

Substances

  • EB3 protein, mouse
  • Microtubule-Associated Proteins
  • Neoplasm Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • cytoplasmic linker protein 170